The present invention provides novel polynucleotides encoding MP-1 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel MP-1 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.
Proteases hydrolyze specific peptide bonds in proteins. The residues at the active site are used to classify proteases (Rawlings and Barrett, 1995). Proteases that hydrolyze peptide bonds using metal ions are referred to as metalloproteases (xe2x80x9cMPxe2x80x9d). The metalloproteinases may be one of the older classes of proteases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures, but many contain a zinc ion. In some cases, zinc may be replaced by another metal such as cobalt or nickel.
The gene of the present invention encodes a human protease belonging to the peptidase m22 family (see Rawlings and Barrett, 1995 for review of protease familial classification). This family contains the sequence HHMEAH (SEQ ID NO:24) The histidine and/or glutamic acid within this sequence are thought to coordinate metal ion binding. Such metal ion coordination facilitates catalysis through the stabilization of a noncovalent, tetrahedral intermediate after the attack of a metal-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group. Metal ion coordination is thought to stabilize the negative charges formed within the active site of the enzyme during catalysis. Such stabilization lowers the transition state energy requirements, and thus results in significant rate enhancements during enzymatic catalysis over non-metal ion coordination conditions (Fersht, A., xe2x80x9cEnzyme Structure and Mechanismxe2x80x9d, 2nd edition, W. H. Freeman and Company, New York, 1985).
The prototype of this family is a secreted O-sialoglycoprotein endopeptidase (so called because it has specificity for cleavage of proteins which contain O-sialoglycans attached to serine and threonine residues) from the bacterium pasteurella haemolytica (Abdullah et al., 1991; Mellors and Lo, 1995). Substrate proteins for the P. haemolytica O-sialoglycoprotein endopeptidase include the cell plasma membrane glycoproteins glycophorin A and leukocyte surface antigens CD34 (expressed on stem cells in the bone marrow), CD43 (a sialomucin implicated in immune cell function and cell signaling), CD44 (a cell receptor for hyalurnate of extracellular matrix) and CD45 (involved in leukocyte activation) (Mellors and Lo, 1995). Other receptors cleaved by this protease include the counter receptor for P-selectin, the counter receptor for L-secton, the receptor for interleukin 7 and epitectin (Mellors and Lo, 1995).
Although the P. haemolytica O-Sialoglycoprotein endopeptidase is the best characterized protease of the peptidase m22 class, genes encoding for other family members have been identified in the genomic sequences of Saccharomyces cerevisiae (baker""s yeast), Borrelia burgdorferi (lyme disease spirochete), Escherichia coli, Helicobacter pylori, Mycobacterium, Haemophilus influenzae and the cyanobacterium Synechocystis. In addition similar sequences have been identified from mycoplasma genitalium, mycoplasma pneumoniae, archaeoglobus fulgidus, pyrococcus horikoshii, chlamydia trachomatis, streptomyces coelicolor, mycobacterium tuberculosis, mycobacterium leprae and bacillus subtilis. The MP-1 gene of the present invention represents the first described mammalian homologue belonging to the m22 class of metalloproteinases.
Metalloproteinases in Disease
Limited-proteolysis by metalloproteases plays a central regulatory role in many physiological and pathophysiological processes. There are many examples of inhibitors of metalloproteases that are useful medications in the treatment of hypertension, heart failure, various forms of cancer and other diseases.
Metalloproteases play many important biological roles in the nervous system, including the spinal cord. There is a balance between the synthesis and degradation of extracellular matrix proteins in the process of synapse formation during development and regeneration. The timing of MP activation is therefore potentially critical. Some MPs have been shown to be upregulated in the spinal cord either during development or in pathological states such as multiple sclerosis, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis. Since MPs degrade extracellular matrix proteins, they would be toxic to developing neurons that depend upon the matrix proteins for survival, neurite outgrowth, and synapse formation. Degradation of the matrix proteins would also cause the breakdown of the blood brain barrier and infiltration of immune cells into the CNS, which occurs in inflammatory conditions such as MS.
Using the above examples, it is clear the availability of a novel cloned metalloproteinase provides opportunities for adjunct or replacement therapy, and are useful for the identification of metalloproteinase agonists, or stimulators (which might stimulate and/or bias metalloproteinase action), as well as, in the identification of metalloproteinase inhibitors. All of which might be therapeutically useful under different circumstances. The metalloproteinase of the present invention can also be used as a scaffold to tailor-make specific metalloproteinase inhibitors.
Polynucleotides and polypeptides corresponding to a portion of the full-length MP-1 polypeptide of the present invention, in addition to its encoding polynucleotides, have been described by Chen et. al. and is described as a putative sialoglycoprotease type 2 protein (Genbank Accession No. gi|11641265).
The protein referenced in Genbank Accession No. gi|11641265 appears to represent an aberrant splice variant of the MP-1 polypeptide of the present invention, having an additional 25 amino acids inserted after position 273 of MP-1 (SEQ ID NO:2). In addition, there are several significant amino acid differences. At amino acid position 31 of SEQ ID NO:2, MP-1 contains a Glycine amino acid (xe2x80x9cGxe2x80x9d) instead of the Glutamic amino acid (xe2x80x9cExe2x80x9d) referenced in Genbank Accession No. gi|11641265. And at amino acid position 373 of SEQ ID NO:2, MP-1 contains an Alanine amino acid (xe2x80x9cAxe2x80x9d), as opposed to the Glycine amino acid (xe2x80x9cGxe2x80x9d) referenced in Genbank Accession No. gi|11641265.
Confirmation that the MP-1 polypeptide sequence of the present invention is the correct sequence, and that the additional 25 amino acids observed within the gi|11641265 protein sequence is a result of aberrant splicing became apparent through the application of several bioinformatics methods. First, an analysis was performed to evaluate the polynucleotide and polypeptide sequence of MP-1 of the present invention to its corresponding genomic sequence (Genbank Accession No. gi|AC013468) as shown in FIG. 6. When AC013468 was analyzed using the GenewiseDB program against MP-1 of the present invention, both the G31 and E373 amino acids were present in the genomic sequence (as shown in FIG. 6). Furthermore, a splice exon/intron splicing junction was clearly present near position 273 of MP-1. In addition, the 25 amino acid insertion observed in gi|11641265 was considered an intron by the GenewiseDB program. When gi|11641265 was analyzed against AC013468 using GenewiseDB, the presence of the 25 amino acid insertion caused a frameshift in the coding sequence. Thus, the 25 amino acid insertion present in gi|11641265 represents an unspliced intron in the cDNA sequence. Thus, based upon the indicia provided above, the MP-1 polypeptide, in addition to, its encoding polynucleotide are believed to represent the physiologically active form of the enzyme.
The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length MP-1 gene and its encoded polypeptide. Also provided are polypeptide alignments illustrating the strong conservation of the MP-1 polypeptide to other known metalloproteinases. Data is also provided illustrating the unique tissue expression profile of the MP-1 polypeptide in spinal cord tissues, which has not been appreciated heretofore.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of MP-1 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MP-1 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the MP-1 protein having the amino acid sequence shown in FIGS. 1A-C (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone, MP-1 (also referred to as protease 3), deposited as ATCC Deposit Number PTA-2766 on Dec. 8, 2000.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of MP-1 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MP-1 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
The invention further provides an isolated MP-1 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention also provides a machine readable storage medium which comprises the structure coordinates of MP-1, including all or any parts conserved metalloproteinase regions. Such storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises said regions or similarly shaped homologous regions.
The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the MP-1 polypeptide. Such compounds are potential inhibitors of MP-1 or its homologues.
The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of MP-1 or its homologues.
The invention also provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises the structural coorrdinates of the model MP-1 in accordance with Table III, or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than about 4.0, 3.0. 2.0, 1.0, or 0.5 angstroms, wherein said computer comprises: A machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the set of structure coordinates of the model MP-1 according to Table III, or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of not more than about 4.0, 3.0. 2.0, 1.0, or 0.5 angstroms; a working memory for storing instructions for processing said machine-readable data; a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and a display coupled to said central-processing unit for displaying said three-dimensional representation.
The invention also provides a computer wherein said machine readable data storage medium is defined by the set of structure coordinates of the model for MP-1 according to Table III, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of not more than about 4.0, 3.0. 2.0, 1.0, or 0.5 angstroms.
The invention also provides a model comprising all or any part of the model defined by structure coordinates of MP-1 according to Table III, or a mutant or homologue of said molecule or molecular complex.
The invention also provides a method for identifying a mutant of MP-1 with altered biological properties, function, or reactivity, the method comprising the step selected from the group consisting of: using the MP-1 model or a homologue of said model according to Table III, for the design of protein mutants with altered biological function or properties which, optionally exhibit the therapeutic effect for MP-1 described herein; and using the MP-1 model or a homologue of said model, for the design of a protein with mutations in the metal binding domain comprised of the amino acids D48, E97, and H146 of SEQ ID NO:2 according to Table III with altered biological function or properties exhibit the therapeutic effect of MP-1 described herein.
The invention also provides a method for identifying modulators of MP-1 biological properties, function, or reactivity, the method comprising the step of modeling test compounds that overlay spatially into the metal binding domain defined by all or any portion of residues D48, E97, and H146, of the three-dimensional MP-1 structural model according to Table III, or using a homologue or portion thereof.
The invention also provides a method for identifying structural and chemical features of MP-1 using the structural coordinates set forth in Table III comprising the step selected from a member of the group consisting of: employing identified structural or chemical features to design or select compounds as potential MP-1 modulators; employing the three-dimensional structural model to design or select compounds as potential MP-1 modulators; synthesizing the potential MP-1 modulators; and screening the potential MP-1 modulators in an assay characterized by binding of a protein to the MP-1.
The invention also provides a method for identifying an MP-1 modulator using the structural coordinates set forth in Table III wherein the potential MP-1 modulator is selected from a database.
The invention also provides a method for identifying an MP-1 modulator using the structural coordinates set forth in Table III wherein the potential MP-1 modulator is designed de novo.
The invention also provides a method for identifying an MP-1 modulator using the structural coordinates set forth in Table III wherein the potential MP-1 modulator is designed from a known modulator of activity.
The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NO:2, or a polypeptide fragment encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.
The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NO:2 or a polypeptide domain encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.
The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NO:2 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.
The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NO:2 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, having biological activity.
The invention further relates to a polynucleotide which is a variant of SEQ ID NO:1.
The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NO:1.
The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NO:2.
The invention further relates to a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:1.
The invention further relates to a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified herein, wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:2, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a metalloproteinase protein.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:2 or the polypeptide encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.
The invention further relates to an isolated nucleic acid molecule of of SEQ ID NO:1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:1 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in the deposited clone.
The invention further relates to a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in the deposited clone, having biological activity.
The invention further relates to a polypeptide domain of SEQ ID NO:2 or the encoded sequence included in the deposited clone.
The invention further relates to a polypeptide epitope of SEQ ID NO:2 or the encoded sequence included in the deposited clone.
The invention further relates to a full length protein of SEQ ID NO:2 or the encoded sequence included in the deposited clone.
The invention further relates to a variant of SEQ ID NO:2.
The invention further relates to an allelic variant of SEQ ID NO:2.
The invention further relates to a species homologue of SEQ ID NO:2.
The invention further relates to the isolated polypeptide of of SEQ ID NO:2, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NO:2.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of SEQ ID NO:2 or the polynucleotide of SEQ ID NO:1.
The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or absence of a mutation in the polynucleotide of SEQ ID NO:1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or amount of expression of the polypeptide of of SEQ ID NO:2 in a biological sample; and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
The invention further relates to a method for identifying a binding partner to the polypeptide of SEQ ID NO:2 comprising the steps of (a) contacting the polypeptide of SEQ ID NO:2 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
The invention further relates to a gene corresponding to the cDNA sequence of SEQ ID NO:1.
The invention further relates to a method of identifying an activity in a biological assay, wherein the method comprises the steps of expressing SEQ ID NO:1 in a cell, (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.
The invention further relates to a process for making polynucleotide sequences encoding gene products having altered SEQ ID NO:2 activity comprising the steps of (a) shuffling a nucleotide sequence of SEQ ID NO:1, (b) expressing the resulting shuffled nucleotide sequences and, (c) selecting for altered activity as compared to the activity of the gene product of said unmodified nucleotide sequence.
The invention further relates to a shuffled polynucleotide sequence produced by a shuffling process, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of SEQ ID NO:2 activity.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a an inflammatory disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a neural disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition an inflammatory disease where proteases, either directly or indirectly, are involved in disease progression.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a cancer.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a blood disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a pulmonary disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a gastrointestinal disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a motor neuron disorder.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is the juvenile form of amyotrophic lateral sclerosis (ALS2).
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is the juvenile form of amyotrophic lateral sclerosis (ALS2).
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is amyotrophic lateral sclerosis (ALS).
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is an amyotrophic lateral sclerosis (ALS)-like condition.
The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is related to aberrant glutamate transport or metabolism.
The invention further relates to a method of identifying a compound that modulates the biological activity of MP-1, comprising the steps of, (a) combining a candidate modulator compound with MP-1 having the sequence set forth in one or more of SEQ ID NO:2; and measuring an effect of the candidate modulator compound on the activity of MP-1.
The invention further relates to a method of identifying a compound that modulates the biological activity of a metalloproteinase, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing MP-1 having the sequence as set forth in SEQ ID NO:2; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed MP-1.
The invention further relates to a method of identifying a compound that modulates the biological activity of MP-1, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein MP-1 is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed MP-1.
The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of MP-1, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of MP-1 in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d) determining the biological activity of MP-1 in the presence of the modulator compound; wherein a difference between the activity of MP-1 in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.
The invention further relates to a compound that modulates the biological activity of human MP-1 as identified by the methods described herein.